There are three myosin V genes in mammals, termed Va, Vb and Vc. Recent studies provide strong evidence that single class V myosin molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, hand over hand, for each diffusional encounter. We demonstrated that the ATPase activity of myosin required calcium for maximal activity and showed that, in the absence of calcium, myosin V adopted a folded, inactive structure. We have used negative staining and cryo-electron microscopy to examine the structure of myosin V that is walking on actin. These images give clear pictures of myosin V molecules with both heads attached to actin and will allow us to make observations about lever arm position and stiffness. Wild type myosin V molecules move processively along actin attached to coverslip surfaces in vitro. The average run length is about 1 um in length. Many investigators have been interested in using myosin V as a motor in nanomachines to move along engineered tracks of actin filaments. In order to increase the run lengths of myosin V, we have cloned cDNA constructs that drive the expression of coiled-coils that form trimeric, tetrameric and pentameric structures fused to the lever arm and motor domain of myosin V. Preliminary data show that we have successfully made a three-headed myosin and that this molecule has a longer run length than seen with the two-headed wild type molecule.